Type II innate lymphoid cell plasticity contributes to impaired reconstitution after allogeneic hematopoietic stem cell transplantation

Type II innate lymphoid cells (ILC2s) maintain homeostasis and barrier integrity in mucosal tissues. In both mice and humans, ILC2s poorly reconstitute after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Determining the mechanisms involved in their impaired reconstitution could improve transplant outcomes. By integrating single-cell chromatin and transcriptomic analyses of transplanted ILC2s, we identify a previously unreported population of converted ILC1-like cells in the mouse small intestine post-transplant. Exposure of ILC2s to proinflammatory cytokines resulted in a mixed ILC1-ILC2 phenotype but was able to convert only a small population of ILC2s to ILC1s, which were found post-transplant. Whereas ILC2s protected against acute graft-versus-host disease (aGVHD) mediated mortality, infusion of proinflammatory cytokine-exposed ILC2s accelerated aGvHD. Interestingly, murine ILC2 reconstitution post-HSCT is decreased in the presence of alloreactive T cells. Finally, peripheral blood cells from human patients with aGvHD have an altered ILC2-associated chromatin landscape compared to transplanted controls. These data demonstrate that following transplantation ILC2s convert to a pro-pathogenic population with an ILC1-like chromatin state and provide insights into the contribution of ILC plasticity to the impaired reconstitution of ILC2 cells, which is one of several potential mechanisms for the poor reconstitution of these important cells after allo-HSCT.

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Life sciences study design
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Sample size
For GvHD experiments, sample sizes were chosen for the effect size needed based on our previous experience of the number of samples needed to demonstrate a significant difference in GvHD scoring between control and treated groups.For the scoring evaluation experiments, the inclusion of 9-12 recipients provided a power of 90% to detect a difference of 14 days in the median GvHD score of > 5 with an alpha error of < 0.05 between control and treated groups.For human experiments (Figure 4), sample size was limited by the number of adults transplanted at the Duke aBMT clinic who could have the time of their sample draws matched to fit the needs of our experimental design.

Replication
For transplant experiments, each group contained a minimum of 6 mice per group and independent experiments were performed a minimum of two times.Biological and technical replicates were both considered wherever possible.For multiomic single cell analysis of ILC2s following transplantation, experiments where cell numbers were severely limited, we were able to prepare libraries and sequence samples in duplicate.
Randomization For in vivo murine experiments, age-matched animals were randomly assigned into control or ILC2 recipient groups.For in vitro cell culture experiments, cells were randomly assigned into different treatment groups.

Blinding
For in vivo experiments we did not have a specific protocol to blind the investigators, however all animals underwent transplantation and cytokine treatment (respectively) at the same time, and blinding was not required for scoring as animals were euthanized prior to the development of fulminant graft versus host disease.For experiments involving human samples, investigators were not blinded to patient groups, however the preparation of single-cell ATAC libraries occurred in two batches, each of which contained the same number of samples from patients with and without aGVHD.

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For sorting experiments: Single cell suspensions were stained with an e450 Lineage antibody cocktail (Invitrogen, 88-7772-72) and BD Horizon AlexaFluor 700 Fixable Viability Stain (BD 564997).Cells were sorted based on GFP expression and GFP+ cells were collected into cR10 prior to downstream processing.

Software
Flow cytometry data were collected with BD FACSDiva software when using a BD analyzer, or with the MACSQuantify software when using the MACSQuant 16 analyzer.All .fcsfiles were analyzed with FlowJo (Version 10.8).

Cell population abundance
A minimum of approximately 10,000 cells were analyzed for any given cytometry sample.Following ex vivo expansion, cell cultures contained 85%+ live ILC2s in both the mouse and human setting.For experiments were FACS sorting was utilized, live, lineage-, GFP+ cells generally represented between 0.5-1% of total cells.

Gating strategy
ILC2s were defined as live (fixable viability stain negative) singlets negative for staining with the Lineage antibody cocktail and positive for ST2/IL-33R (mouse) or CD127 (human).Upon gating for single, viable ILC2s, fluorescence minus one (FMO) or controls were used to establish negative and positive gates.In the case of cells that underwent PMA and ionomycin stimulation for intracellular cytokine or transcription factor staining, unstimulated cells (treated with Brefeldin A alone) were reserved as a negative control.All experiments included unstained and single color compensation controls.
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